Chapter 20

1. Chapter 20 DNA Technology and Genomics

2. Overview: Understanding and Manipulating Genomes • Sequencing of the human genome was largely completed by 2003 • DNA sequencing has depended on advances in technology, starting with making recombinant DNA • In recombinant DNA, nucleotide sequences from two different sources, often two species, are combined in vitro into the same DNA molecule • Methods for making recombinant DNA are central to genetic engineering, the direct manipulation of genes for practical purposes

3. DNA technology has revolutionized biotechnology, the manipulation of organisms or their genetic components to make useful products

4. Concept 20.1: DNA cloning permits production of multiple copies of a specific gene or other DNA segment • To work directly with specific genes, scientists prepare gene-sized pieces of DNA in identical copies, a process called gene cloning

5. DNA Cloning and Its Applications: A Preview • Most methods for cloning pieces of DNA in the laboratory share general features, such as the use of bacteria and their plasmids • Cloned genes are useful for making copies of a particular gene and producing a gene product

6. Cell containing gene of interest Bacterium Gene inserted into plasmid LE 20-2 Bacterial chromosome Plasmid Gene of interest Recombinant DNA (plasmid) DNA of chromosome Plasmid put into bacterial cell Recombinant bacterium Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Protein expressed by gene of interest Gene of interest Copies of gene Protein harvested Basic research and various applications Basic research on gene Basic research on protein Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Human growth hor- mone treats stunted growth

7. Using Restriction Enzymes to Make Recombinant DNA • Bacterial restriction enzymes cut DNA molecules at DNA sequences called restriction sites • A restriction enzyme usually makes many cuts, yielding restriction fragments • The most useful restriction enzymes cut DNA in a staggered way, producing fragments with “sticky ends” that bond with complementary “sticky ends” of other fragments • DNA ligase is an enzyme that seals the bonds between restriction fragments

8. Restriction site 5¢ 3¢ DNA 3¢ 5¢ LE 20-3 Restriction enzyme cuts the sugar-phosphate backbones at each arrow. Sticky end DNA fragment from another source is added. Base pairing of sticky ends produces various combinations. Fragment from different DNA molecule cut by the same restriction enzyme One possible combination DNA ligase seals the strands. Recombinant DNA molecule

9. Animation: Restriction Enzymes

10. Cloning a Eukaryotic Gene in a Bacterial Plasmid • In gene cloning, the original plasmid is called a cloning vector • A cloning vector is a DNA molecule that can carry foreign DNA into a cell and replicate there

11. Producing Clones of Cells • Cloning a human gene in a bacterial plasmid can be divided into six steps: 1. Vector and gene-source DNA are isolated 2. DNA is inserted into the vector 3. Human DNA fragments are mixed with cut plasmids, and base-pairing takes place 4. Recombinant plasmids are mixed with bacteria 5. The bacteria are plated and incubated 6. Cell clones with the right gene are identified Animation: Cloning a Gene

12. Bacterial cell lacZ gene (lactose breakdown) Human cell LE 20-4_1 Isolate plasmid DNA and human DNA. Restriction site ampR gene (ampicillin resistance) Bacterial plasmid Gene of interest Sticky ends Human DNA fragments Cut both DNA samples with the same restriction enzyme. Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids. Recombinant DNA plasmids

13. Bacterial cell lacZ gene (lactose breakdown) Human cell Isolate plasmid DNA and human DNA. LE 20-4_2 Restriction site ampR gene (ampicillin resistance) Bacterial plasmid Gene of interest Sticky ends Human DNA fragments Cut both DNA samples with the same restriction enzyme. Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids. Recombinant DNA plasmids Introduce the DNA into bacterial cells that have a mutation in their own lacZ gene. Recombinant bacteria

14. lacZ gene (lactose breakdown) Bacterial cell Human cell Isolate plasmid DNA and human DNA. Restriction site LE 20-4_3 ampR gene (ampicillin resistance) Bacterial plasmid Gene of interest Sticky ends Human DNA fragments Cut both DNA samples with the same restriction enzyme. Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids. Recombinant DNA plasmids Introduce the DNA into bacterial cells that have a mutation in their own lacZ gene. Recombinant bacteria Plate the bacteria on agar containing ampicillin and X-gal. Incubate until colonies grow. Colony carrying recombinant plasmid with disrupted lacZ gene Colony carrying non- recombinant plasmid with intact lacZ gene Bacterial clone

15. Identifying Clones Carrying a Gene of Interest • A clone carrying the gene of interest can be identified with a nucleic acid probe having a sequence complementary to the gene • This process is called nucleic acid hybridization • An essential step in this process is denaturation of the cells’ DNA, separation of its two strands

16. Colonies containing gene of interest LE 20-5 Master plate Master plate Probe DNA Radioactive single-stranded DNA Solution containing probe Gene of interest Film Single-stranded DNA from cell Filter Filter lifted and flipped over Hybridization on filter A special filter paper is pressed against the master plate, transferring cells to the bottom side of the filter. The filter is treated to break open the cells and denature their DNA; the resulting single-stranded DNA molecules are treated so that they stick to the filter. The filter is laid under photographic film, allowing any radioactive areas to expose the film (autoradiography). After the developed film is flipped over, the reference marks on the film and master plate are aligned to locate colonies carrying the gene of interest.

17. Bacterial Expression Systems • Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells • To overcome differences in promoters and other DNA control sequences, scientists usually employ an expression vector, a cloning vector that contains a highly active prokaryotic promoter

18. One method of introducing recombinant DNA into eukaryotic cells is electroporation, applying a brief electrical pulse to create temporary holes in plasma membranes • Alternatively, scientists can inject DNA into cells using microscopic needles • Once inside the cell, the DNA is incorporated into the cell’s DNA by natural genetic recombination

19. Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR) • The polymerase chain reaction, PCR, can produce many copies of a specific target segment of DNA • A three-step cycle—heating, cooling, and replication—brings about a chain reaction that produces an exponentially growing population of identical DNA molecules

20. 5¢ 3¢ Target sequence Genomic DNA 3¢ 5¢ LE 20-7 5¢ 3¢ Denaturation: Heat briefly to separate DNA strands 3¢ 5¢ Annealing: Cool to allow primers to form hydrogen bonds with ends of target sequence Cycle 1 yields 2 molecules Primers Extension: DNA polymerase adds nucleotides to the 3¢ end of each primer New nucleo- tides Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence

21. Concept 20.2: Restriction fragment analysis detects DNA differences that affect restriction sites • Restriction fragment analysis detects differences in the nucleotide sequences of DNA molecules • Such analysis can rapidly provide comparative information about DNA sequences

22. Gel Electrophoresis and Southern Blotting • One indirect method of rapidly analyzing and comparing genomes is gel electrophoresis • This technique uses a gel as a molecular sieve to separate nuclei acids or proteins by size Video: Biotechnology Lab

23. Mixture of DNA molecules of differ- ent sizes Longer molecules LE 20-8 Cathode Shorter molecules Power source Gel Glass plates Anode

24. In restriction fragment analysis, DNA fragments produced by restriction enzyme digestion of a DNA molecule are sorted by gel electrophoresis • Restriction fragment analysis is useful for comparing two different DNA molecules, such as two alleles for a gene

25. Normal b-globin allele 175 bp 201 bp Large fragment LE 20-9 Ddel Ddel Ddel Ddel Sickle-cell mutant b-globin allele 376 bp Large fragment Ddel Ddel Ddel Ddel restriction sites in normal and sickle-cell alleles of -globin gene Normal allele Sickle-cell allele Large fragment 376 bp 201 bp 175 bp Electrophoresis of restriction fragments from normal and sickle-cell alleles

26. A technique called Southern blotting combines gel electrophoresis with nucleic acid hybridization • Specific DNA fragments can be identified by Southern blotting, using labeled probes that hybridize to the DNA immobilized on a “blot” of gel

27. Heavy weight Restriction fragments DNA + restriction enzyme Nitrocellulose paper (blot) I I I Gel LE 20-10 Sponge Paper towels I Normal -globin allele I Sickle-cell allele I Heterozygote Alkaline solution Preparation of restriction fragments. Gel electrophoresis. Blotting. Probe hydrogen- bonds to fragments containing normal or mutant -globin I I I Radioactively labeled probe for -globin gene is added to solution in a plastic bag I I I Fragment from sickle-cell -globin allele Film over paper blot Fragment from normal -globin allele Paper blot Hybridization with radioactive probe. Autoradiography.

28. Restriction Fragment Length Differences as Genetic Markers • Restriction fragment length polymorphisms (RFLPs, or Rif-lips) are differences in DNA sequences on homologous chromosomes that result in restriction fragments of different lengths • A RFLP can serve as a genetic marker for a particular location (locus) in the genome • RFLPs are detected by Southern blotting

29. Concept 20.3: Entire genomes can be mapped at the DNA level • The most ambitious mapping project to date has been the sequencing of the human genome • Officially begun as the Human Genome Project in 1990, the sequencing was largely completed by 2003 • Scientists have also sequenced genomes of other organisms, providing insights of general biological significance • Go to video

30. Genetic (Linkage) Mapping: Relative Ordering of Markers • The first stage in mapping a large genome is constructing a linkage map of several thousand genetic markers throughout each chromosome • The order of markers and relative distances between them are based on recombination frequencies

31. Chromosome bands Cytogenetic map Genes located by FISH LE 20-11 Genetic (linkage) mapping Genetic markers Physical mapping Overlapping fragments DNA sequencing

32. Physical Mapping: Ordering DNA Fragments • A physical map is constructed by cutting a DNA molecule into many short fragments and arranging them in order by identifying overlaps • Physical mapping gives the actual distance in base pairs between markers

33. DNA Sequencing • Relatively short DNA fragments can be sequenced by the dideoxy chain-termination method • Inclusion of special dideoxyribonucleotides in the reaction mix ensures that fragments of various lengths will be synthesized

34. DNA (template strand) Primer Deoxyribonucleotides Dideoxyribonucleotides (fluorescently tagged) 3¢ 5¢ 5¢ LE 20-12 DNA polymerase 3¢ DNA (template strand) Labeled strands 3¢ 5¢ 3¢ Direction of movement of strands Laser Detector

35. Linkage mapping, physical mapping, and DNA sequencing represent the overarching strategy of the Human Genome Project • An alternative approach to sequencing genomes starts with sequencing random DNA fragments • Computer programs then assemble overlapping short sequences into one continuous sequence

36. Cut the DNA from many copies of an entire chromosome into overlapping frag-ments short enough for sequencing LE 20-13 Clone the fragments in plasmid or phage vectors Sequence each fragment Order the sequences into one overall sequence with computer software

37. Concept 20.4: Genome sequences provide clues to important biological questions • In genomics, scientists study whole sets of genes and their interactions • Genomics is yielding new insights into genome organization, regulation of gene expression, growth and development, and evolution

38. Identifying Protein-Coding Genes in DNA Sequences • Computer analysis of genome sequences helps identify sequences likely to encode proteins • The human genome contains about 25,000 genes, but the number of human proteins is much larger • Comparison of sequences of “new” genes with those of known genes in other species may help identify new genes

40. Determining Gene Function • One way to determine function is to disable the gene and observe the consequences • Using in vitro mutagenesis, mutations are introduced into a cloned gene, altering or destroying its function • When the mutated gene is returned to the cell, the normal gene’s function might be determined by examining the mutant’s phenotype • In nonmammalian organisms, a simpler and faster method, RNA interference (RNAi), has been used to silence expression of selected genes

41. Studying Expression of Interacting Groups of Genes • Automation has allowed scientists to measure expression of thousands of genes at one time using DNA microarray assays • DNA microarray assays compare patterns of gene expression in different tissues, at different times, or under different conditions

42. Tissue sample Isolate mRNA. LE 20-14 mRNA molecules Make cDNA by reverse transcription, using fluorescently labeled nucleotides. Apply the cDNA mixture to a microarray, a microscope slide on which copies of single-stranded DNA fragments from the organism’s genes are fixed, a different gene in each spot. The cDNA hybridizes with any complementary DNA on the microarray. Labeled cDNA molecules (single strands) DNA microarray Rinse off excess cDNA; scan microarray for fluorescent. Each fluorescent spot (yellow) represents a gene expressed in the tissue sample. Size of an actual DNA microarray with all the genes of yeast (6,400 spots)

43. Comparing Genomes of Different Species • Comparative studies of genomes from related and widely divergent species provide information in many fields of biology • The more similar the nucleotide sequences between two species, the more closely related these species are in their evolutionary history • Comparative genome studies confirm the relevance of research on simpler organisms to understanding human biology

44. Future Directions in Genomics • Genomics is the study of entire genomes • Proteomics is the systematic study of all proteins encoded by a genome • Single nucleotide polymorphisms (SNPs) provide markers for studying human genetic variation

45. Concept 20.5: The practical applications of DNA technology affect our lives in many ways • Many fields benefit from DNA technology and genetic engineering

46. Medical Applications • One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases

47. Diagnosis of Diseases • Scientists can diagnose many human genetic disorders by using PCR and primers corresponding to cloned disease genes, then sequencing the amplified product to look for the disease-causing mutation • Even when a disease gene has not been cloned, presence of an abnormal allele can be diagnosed if a closely linked RFLP marker has been found

48. LE 20-15 RFLP marker DNA Disease-causing allele Restriction sites Normal allele

49. Human Gene Therapy • Gene therapy is the alteration of an afflicted individual’s genes • Gene therapy holds great potential for treating disorders traceable to a single defective gene • Vectors are used for delivery of genes into cells • Gene therapy raises ethical questions, such as whether human germ-line cells should be treated to correct the defect in future generations

50. Cloned gene Insert RNA version of normal allele into retrovirus. LE 20-16 Viral RNA Let retrovirus infect bone marrow cells that have been removed from the patient and cultured. Retrovirus capsid Viral DNA carrying the normal allele inserts into chromosome. Bone marrow cell from patient Bone marrow Inject engineered cells into patient.